Secondary metabolites in Gerbera hybrida

Plants produce a diversity of secondary metabolites, i.e., low-molecular-weight compounds that have primarily ecological functions in plants. The flavonoid pathway is one of the most studied biosynthetic pathways in plants. In order to understand biosynthetic pathways fully, it is necessary to isolate and purify the enzymes of the pathways to study individual steps and to study the regulatory genes of the pathways. Chalcone synthases are key enzymes in the formation of several groups of flavonoids, including anthocyanins. In this study, a new chalcone synthase enzyme (GCHS4), which may be one of the main contributors to flower colour, was characterised from the ornamental plant Gerbera hybrida. In addition, four chalcone synthase-like genes and enzymes (GCHS17, GCHS17b, GCHS26 and GCHS26b) were studied. Spatial expression of the polyketide synthase gene family in gerbera was also analysed with quantitative RT-PCR from 12 tissues, including several developmental stages and flower types. A previously identified MYB transcription factor from gerbera, GMYB10, which regulates the anthocyanin pathway, was transferred to gerbera and the phenotypes were analysed. Total anthocyanin content and anthocyanidin profiles of control and transgenic samples were compared spectrophotometrically and with HPLC. The overexpression of GMYB10 alone was able to change anthocyanin pigmentation: cyanidin pigmentation was induced and pelargonidin pigmentation was increased. The gerbera 9K cDNA microarray was used to compare the gene expression profiles of transgenic tissues against the corresponding control tissues to reveal putative target genes for GMYB10. GMYB10 overexpression affected the expression of both early and late biosynthetic genes in anthocyanin-accumulating transgenic tissues, including the newly isolated gene GCHS4. Two new MYB domain factors, named as GMYB11 and GMYB12, were also upregulated. Gene transfer is not only a powerful tool for basic research, but also for plant breeding. However, crop improvement by genetic modification (GM) remains controversial, at least in Europe. Many of the concerns relating to both human health and to ecological impacts relate to changes in the secondary metabolites of GM crops. In the second part of this study, qualitative and quantitative differences in cytotoxicity and metabolic fingerprints between 225 genetically modified Gerbera hybrida lines and 42 non-GM Gerbera varieties were compared. There was no evidence for any major qualitative and quantitative changes between the GM lines and non-GM varieties. The developed cell viability assays offer also a model scheme for cell-based cytotoxicity screening of a large variety of GM plants in standardized conditions.

Novel Molecular Mechanisms of Arabidopsis Disease Resistance

Plants are capable of recognizing phytopathogens through the perception of pathogen-derived molecules or plant cell-wall degradation products due to the activities of pathogen-secreted enzymes. Such elicitor recognition events trigger an array of inducible defense responses involving signal transduction networks and massive transcriptional re-programming. The outcome of a pathogen infection relies on the balance between different signaling pathways, which are integrated by regulatory proteins. This thesis characterized two key regulatory components: a damage control enzyme, chlorophyllase 1 (AtCHL1), and a transcription factor, WRKY70. Their roles in defense signaling were then investigated. The Erwinia-derived elicitors rapidly activated the expression of AtCLH1 and WRKY70 through different signaling pathways. The expression of the AtCHL1 gene was up-regulated by jasmonic acid (JA) but down-regulated by salicylic acid (SA), whereas WRKY70 was activated by SA and repressed by JA. In order to elucidate the functions of AtCLH1 and WRKY70 in plant defense, stable transgenic lines were produced where these genes were overexpressed or silenced. Additionally, independent knockout lines were also characterized. Bacterial and fungal pathogens were then used to assess the contribution of these genes to the Arabidopsis disease resistance. The transcriptional modulation of AtCLH1 by either the constitutive over-expression or RNAi silencing caused alterations in the chlorophyll-to-chlorophyllide ratio, supporting the claim that chlorophyllase 1 has a role in the chlorophyll degradation pathway. Silencing of this gene led to light-dependent over-accumulation of the reactive oxygen species (ROS) in response to infection by Erwinia carotovora subsp. carotovora SCC1. This was followed by an enhanced induction of SA-dependent defense genes and an increased resistance to this pathogen. Interestingly, little effect on the pathogen-induced SA accumulation at the early infection was observed, suggesting that action of ROS might potentiate SA signaling. In contrast, the pathogen-induced JA production was significantly reduced in the RNAi silenced plants. Moreover, JA signaling and resistance to Alternaria brassicicola were impaired. These observations provide support for the argument that the ROS generated in chloroplasts might have a negative impact on JA signaling. The over-expression of WRKY70 resulted in an enhanced resistance to E. carotovora subsp. carotovora SCC1, Pseudomonas syringae pv. tomato DC3000 and Erysiphe cichoracearum UCSC1, whilst an antisense suppression or an insertional inactivation of WRKY70 led to a compromised resistance to E. carotovora subsp. carotovora SCC1 and to E. cichoracearum UCSC1 but not to P. syringae pv. tomato DC3000. Gene expression analysis revealed that WRKY70 activated many known defense-related genes associated with the SAR response but suppressed a subset of the JA-responsive genes. In particular, I was able to show that both the basal and the induced expression of AtCLH1 was enhanced by the antisense silencing or the insertional inactivation of WRKY70, whereas a reduction in AtCLH1 expression was observed in the WRKY70 over-expressors following an MeJA application or an A. brassicicola infection. Moreover, the SA-induced suppression of AtCLH1 was relieved in wrky70 mutants. These results indicate that WRKY70 down-regulates AtCLH1. An epistasis analysis suggested that WRKY70 functions downstream of the NPR1 in an SA-dependent signaling pathway. When challenged with A. brassicicola, WRKY70 over-expressing plants exhibited a compromised disease resistance while wrky70 mutants had the opposite effect. These results confirmed the WRKY70-mediated inhibitory effects on JA signaling. Furthermore, the WRKY70-controlled suppression of A. brassicicola resistance was mainly through an NPR1-dependent mechanism. Taking all the data together, I suggest that the pathogen-responsive transcription factor WRKY70 is a common component in both SA- and JA-dependent pathways and plays a crucial role in the SA-mediated suppression of JA signaling.

Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80A degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 A mu g/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.

Proteomic identification of putative microRNA394 target genes in Arabidopsis thaliana identifies MLP family members critical for normal development

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem (SAM) organisation. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Major Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR. Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development.

Kirjallisuuskatsaus: Eturauhassyövän androgeeniresponsiiviset in vivo -mallit; Kokeellinen osa: Bioluminesenssiin perustuvan ortotooppisen eturauhassyöpämallin optimointi

Eturauhassyöpä on yksi yleisimmistä syövistä länsimaissa. Eturauhassyöpä on yleensä hitaasti kehittyvä tauti. Edetessään se voi kuitenkin muuntua aggressiivisemmaksi ja aiheuttaa metastaaseja, jotka ovat pääasiallisena syynä taudin kuolleisuuteen. Androgeenit ovat merkittäviä tekijöitä eturauhassyövän patogeneesissä ja eturauhassyöpäkudos on useimmiten riippuvainen androgeeneista. Tämän vuoksi hoidon tavoitteena on estää niiden eritys kirurgisella tai kemiallisella kastraatiolla ja/tai estää androgeenien vaikutus antiandrogeeneilla. Eturauhassyöpää sekä sen hoitoon tarkoitettuja uusia lääkehoitomahdollisuuksia tutkitaan kiivaasti. Eturauhassyövän tutkimiseen on kehitetty lukematon määrä erilaisia in vivo -malleja. Koska eturauhassyöpä on yleensä androgeeneille herkkä, kuvaavat androgeeniresponsiiviset eläinmallit ihmisen tautia parhaiten. Eturauhassyövän mallintamiseen in vivo voidaan käyttää eri eläinlajeja, mutta hiiri on ylivoimaisesti käytetyin mallieläin. Immuunipuutteisiin hiiriin voidaan aiheuttaa kasvaimia inokuloimalla ihmisen kasvainsoluja tai osia ihmisen kasvaimista. Ortotooppisesti eturauhaseen inokuloitavat kasvainmallit mallintavat eturauhassyövässä esiintyvää syöpäsolujen ja stroomasolujen välistä epänormaalia vuorovaikutusta. Muuntogeeniset hiirimallit ovat yhä yleisempiä eturauhassyövän tutkimuksessa. Muuntogeenisilla malleilla voidaan mallintaa taudin kehittymistä ja sen etenemistä kokonaisuudessaan parhaiten. Eturauhasessa olevaa kasvainta ja sen kasvua on vaikea seurata ilman prostataspesifisen antigeenin (PSA) pitoisuuden mittausta tai erityisiä kuvantamistekniikoita. Tällaisia menetelmiä, kuten optista kuvantamista, käytetään yhä enemmän hyödyksi erilaisissa eturauhassyövän in vivo -malleissa. Tutkielman kokeellisen osan tavoitteena oli optimoida bioluminesenssiin perustuva optinen kuvantamismenetelmä androgeeniresponsiivisessa LNCaP-luc2-solulinjassa ortotooppisessa eturauhassyöpämallissa. Bioluminesenssikuvantaminen perustuu kasvainsolujen ilmentämän lusiferaasin katalysoimaan reaktioon, jossa entsyymin substraatti, lusiferiini, hapettuu ja tuottaa näkyvää valoa. Lisäksi tavoitteena oli tutkia lääkehoitojen ja kastraation vasteita mallissa. Bioluminesenssiin perustuvalla kuvantamisella oli mahdollista seurata eturauhaskasvainten kasvua noninvasiivisesti, reaaliaikaisesti ja toistuvasti. Bioluminesenssikuvantamisen avulla kasvainten kvantitointi oli nopeampaa kuin ultraäänikuvantamisen avulla, ja kasvainten kasvua oli myös mahdollista seurata useammin kuin seerumin PSA-mittausten avulla. Bioluminesenssikuvantamisen todettiin korreloivan paremmin PSA-pitoisuuden kanssa kuin kasvaimen todelliseen kokoon lopetushetkellä. Seerumin PSA-pitoisuus korreloi kuitenkin bioluminesenssimittausta paremmin eturauhaskasvaimen kokoon tässä kokeessa. Kasvainten oletettua suurempaa kokoa voidaan pitää todennäköisimpänä syynä sille, ettei lääkehoitojen tai kastraation todettu vaikuttavan kasvainten kasvuun bioluminesenssikuvantamisella mitattuna. Bioluminesenssikuvantaminen ei sovellu suurille eikä nekroottisille kasvaimille, sillä kuvantamismenetelmä toimii vain elävillä soluilla. Bioluminesenssikuvantamisen hyödyntämisen kannalta oleellista tässä mallissa on myös lusiferiini-injektion onnistuminen. Jatkotutkimuksia tarvitaan edelleen mallin validoimiseksi mm. lääkehoitojen vasteiden osoittamiseksi.

Transfer of saline tolerance from one strain of rice to another by injection of DNA

Direct injection of genomic DNA from salt tolerant cv. Pokkali into developing floral tillers on IR20 produced transgenic seeds similar to Pokkali in husk colour and which germinated well in 0.2 M NaCl and had a 4-6-fold higher proline content.

Biphenyl ethers conjugated CdSe/ZnS core/shell quantum dots and interpretation of the mechanism of amyloid fibril disruption

The biphenyl ethers (BPEs) are the potent inhibitors of TTR fibril formation and are efficient fibril disrupter. However, the mechanism by which the fibril disruption occurs is yet to be fully elucidated. To gain insight into the mechanism, we synthesized and used a new QD labeled BPE to track the process of fibril disruption. Our studies showed that the new BPE-QDs bind to the fiber uniformly and has affinity and specificity for TTR fiber and disrupted the pre-formed fiber at a relatively slow rate. Based on these studies we put forth the probable mechanism of fiber disruption by BPEs. Also, we show here that the BPE-QDs interact with high affinity to the amyloids of A beta(42), lysozyme and insulin. The potential of BPE-QDs in the detection of senile plaque in the brain of transgenic Alzheimer's mice has also been explored. (C) 2010 Elsevier Ltd. All rights reserved.

Molecular mechanisms of hypertension-induced heart failure : Experimental studies with special emphasis on local renin-angiotensin system, cardiac metabolism and levosimendan

Hypertension is a major risk factor for stroke, ischaemic heart disease, and the development of heart failure. Hypertension-induced heart failure is usually preceded by the development of left ventricular hypertrophy (LVH), which represents an adaptive and compensatory response to the increased cardiac workload. Biomechanical stress and neurohumoral activation are the most important triggers of pathologic hypertrophy and the transition of cardiac hypertrophy to heart failure. Non-clinical and clinical studies have also revealed derangements of energy metabolism in hypertensive heart failure. The goal of this study was to investigate in experimental models the molecular mechanisms and signalling pathways involved in hypertension-induced heart failure with special emphasis on local renin-angiotensin-aldosterone system (RAAS), cardiac metabolism, and calcium sensitizers, a novel class of inotropic agents used currently in the treatment of acute decompensated heart failure. Two different animal models of hypertensive heart failure were used in the present study, i.e. hypertensive and salt-sensitive Dahl/Rapp rats on a high salt diet (a salt-sensitive model of hypertensive heart failure) and double transgenic rats (dTGR) harboring human renin and human angiotensinogen genes (a transgenic model of hypertensive heart failure with increased local RAAS activity). The influence of angiotensin II (Ang II) on cardiac substrate utilization and cardiac metabolomic profile was investigated by using gas chromatography coupled to time-of-flight mass spectrometry to detect 247 intermediary metabolites. It was found that Ang II could alter cardiac metabolomics both in normotensive and hypertensive rats in an Ang II receptor type 1 (AT1)-dependent manner. A distinct substrate use from fatty acid oxidation towards glycolysis was found in dTGR. Altered cardiac substrate utilization in dTGR was associated with mitochondrial dysfunction. Cardiac expression of the redox-sensitive metabolic sensor sirtuin1 (SIRT1) was increased in dTGR. Resveratrol supplementation prevented cardiovascular mortality and ameliorated Ang II-induced cardiac remodeling in dTGR via blood pressure-dependent pathways and mechanisms linked to increased mitochondrial biogenesis. Resveratrol dose-dependently increased SIRT1 activity in vitro. Oral levosimendan treatment was also found to improve survival and systolic function in dTGR via blood pressure-independent mechanisms, and ameliorate Ang II-induced coronary and cardiomyocyte damage. Finally, using Dahl/Rapp rats it was demonstrated that oral levosimendan as well as the AT1 receptor antagonist valsartan improved survival and prevented cardiac remodeling. The beneficial effects of levosimendan were associated with improved diastolic function without significantly improved systolic changes. These positive effects were potentiated when the drug combination was administered. In conclusion, the present study points to an important role for local RAAS in the pathophysiology of hypertension-induced heart failure as well as its involvement as a regulator of cardiac substrate utilization and mitochondrial function. Our findings suggest a therapeutic role for natural polyphenol resveratrol and calcium sensitizer, levosimendan, and the novel drug combination of valsartan and levosimendan, in prevention of hypertension-induced heart failure. The present study also provides a better understanding of the pathophysiology of hypertension-induced heart failure, and may help identify potential targets for novel therapeutic interventions.

Bovine mastitis caused by coagulase-negative staphylococci : host response and bacterial factors

Coagulase-negative staphylococci (CNS) are the most common bacteria isolated in bovine subclinical mastitis in many countries, and also a frequent cause of clinical mastitis. The most common species isolated are Staphylococcus (S) chromogenes, S. simulans, S. epidermidis, and S. xylosus. One half of the intramammary infections (IMI) caused by CNS persist in the udder. The pathogenesis of IMI caused by CNS is poorly understood. This dissertation focuses on host response in experimental intramammary infection induced by S. chromogenes, S. epidermidis and S. simulans. Model for a mild experimental CNS infection was developed with S. chromogenes (study I). All cows were infected and most developed subclinical mastitis. In study II the innate immune response to S. epidermidis and S. simulans IMI was compared in eight cows using a crossover design. A larger dose of bacteria was used to induce clinical mastitis. All cows became infected and showed mild to moderate clinical signs of mastitis. S. simulans caused a slightly stronger innate immune response than S. epidermidis, with significantly higher concentrations of the interleukins IL-1beta and IL-8 in the milk. The spontaneous elimination rate of the 16 IMIs was 31%, with no difference between species. No significant differences were recorded between infections eliminated spontaneously or remaining persistent, although the response was stronger in IMIs eliminated spontaneously, except the concentration of TNF-α, which remained elevated in persistent infections. Lactoferrin (Lf) is a component of the humoral defence of the host and is present at low concentrations in the milk. The concentration of Lf in milk is high during the dry period, in colostrum, and in mastitic milk. The effect of an inherent, high concentration of Lf in the milk on experimental IMI induced with S. chromogenes was studied in transgenic cows that expressed recombinant human Lf in their milk. Human Lf did not prevent S. chromogenes IMI, but the host response was milder in transgenic cows than in normal cows, and the former eliminated infection faster. Biofilm production has been suggested to promote persistence of IMI. Phenotypic biofilm formation and slime producing ability of CNS isolates from bovine mastitis was investigated in vitro. One-third of mastitis isolates produced biofilm. Slime production was less frequent for isolates of the most common mastitis causing species S. chromogenes and S. simulans compared with S. epidermidis. No association was found between the phenotypic ability to form biofilm and the persistence of IMI or severity of mastitis. Slime production was associated with persistent infections, but only 8% of isolates produced slime.

Kirjallisuusosa: Alzheimerin taudin rottamallien vastaavuus tautiin ihmisessä ja mallien käyttökelpoisuus

Kirjallisuusosa: Alzheimerin taudin hoitoon olisi tarvetta uusille taudinkulkuun vaikuttaville lääkeaineille. Niiden kehittämiseksi tarvitaan eläinmalleja, joissa esiintyy taudin patofysiologisia piirteitä. Rottamalleista vanhemmat skopolamiini- tai MK-801-häirintä sekä ikääntyneiden rottien käyttö eivät kovin hyvin vastaa taudin patofysiologiaa, vaikka niissä eläimen muisti käyttäytymiskokeissa onkin heikentynyt. Uudemmat transgeeniset rottamallit ja mallit, joissa annetaan Aβ:a aivoihin, ilmentävät huomattavasti paremmin Alzheimerin taudin kaltaista tilaa aivoissa ainakin Aβ:n osalta. Taupatofysiologiaa ei silti kummassakaan näistä malleista juuri esiinny. Toisaalta Aβ:lla näyttäisi olevan huomattavasti tau:ta suurempi rooli taudissa, joten sen ilmeneminen mallissa onkin keskeisempi tekijä. Nämä mallit ilmentävät melko suurelti osin yhtä hyvin Alzheimerin taudin patofysiologiaa. Aβ:n antaminen on hieman yksinkertaisempi suorittaa käytännössä, sillä siinä ei tarvitse luoda transgeenista kantaa. Toisaalta transgeenisessa mallissa Aβ-patofysiologia syntyy enemmän Alzheimerin taudin kaltaisesti solujen sisällä eikä valmiita aggregoituvia Aβ-peptidejä anneta ulkopuolelta aivoihin. Molemmat mallit ovat kuitenkin käyttökelpoisia, ja soveltuvat erityisesti Aβ:an vaikuttavien lääkeaineiden kehittämiseen. Kokeellinen osa: Kokeen tarkoituksena oli validoida kohotettu ristikko-sokkelo (elevated plus-maze, EPM) hiirillä kognitiomallina. Kokeessa käytettiin kahden koekerran (trial, T) menetelmää, jossa koekertojen pituus oli viisi minuuttia. Näin saatiin useita oppimista kuvaavia parametreja. Hiirille yritettiin saada muistihäiriö aikaviiveen avulla (koekertojen väli 1-18 vrk) tai antamalla muskariinireseptoriantagonistia skopolamiinia (0,1-0,8 mg/kg i.p.) 30 minuuttia ennen T1:tä. Nämä kokeet suoritettiin sekä C57BL/6J- että ICR:(CD-1)-hiirillä. Aikaviivekokeissa ainut ryhmä, jolla oli viitettä unohtamisesta, oli ICR:(CD-1)-hiirien 18 vrk:n ryhmä. Tämän perusteella tutkittiin vielä 21 vuorokauden aikaväli, mutta selvää muistihäiriötä ei esiintynyt. Skopolamiini ei häirinnyt muistia ICR:(CD-1)-hiirillä, mutta C57BL/6J-hiirillä 0,2 mg/kg:n annoksesta ylöspäin merkitsevä muistihäiriö esiintyi. Näin ollen jatkokokeissa käytettäväksi valittiin skopolamiinin annos 0,2 mg/kg C57BL/6J-hiirillä, ja siinä tutkittiin donepetsiilin (0,3, 0,8 ja 1,5 mg/kg s.c), memantiinin (5,0 ja 10,0 mg/kg s.c) ja kokeellisen 5-HT6-antagonistin SB742457:n (1,5 ja 6,0 mg/kg s.c) muistia parantavia vaikutuksia. Tutkittavat lääkeaineet annettiin 40 minuuttia ennen T1:tä ja skopolamiini 30 minuuttia ennen. Memantiinilla (5,0 mg/kg) oli selkeä skopolamiinin heikentämää kognitiota parantava vaikutus ja donepetsiilillakin (1,5 mg/kg) suuntaus tähän. Tulosten perusteella malli näyttäisi soveltuvan muisti- ja oppimisvaikutusten tutkimiseen käytettäväksi malliksi.

LIN28B, LIN28A, KISS1, and KISS1R in idiopathic central precocious puberty

Abstract Background Pubertal timing is a strongly heritable trait, but no single puberty gene has been identified. Thus, the genetic background of idiopathic central precocious puberty (ICPP) is poorly understood. Overall, the genetic modulation of pubertal onset most likely arises from the additive effect of multiple genes, but also monogenic causes of ICPP probably exist, as cases of familial ICPP have been reported. Mutations in KISS1 and KISSR, coding for kisspeptin and its receptor, involved in GnRH secretion and puberty onset, have been suggested causative for monogenic ICPP. Variation in LIN28B was associated with timing of puberty in genome-wide association (GWA) studies. LIN28B is a human ortholog of the gene that controls, through microRNAs, developmental timing in C. elegans. In addition, Lin28a transgenic mice manifest the puberty phenotypes identified in the human GWAS. Thus, both LIN28B and LIN28A may have a role in pubertal development and are good candidate genes for monogenic ICPP. Methods Thirty girls with ICPP were included in the study. ICPP was defined by pubertal onset before 8 yrs of age, and a pubertal LH response to GnRH testing. The coding regions of LIN28B, LIN28A, KISS1, and KISS1R were sequenced. The missense change in LIN28B was also screened in 132 control subjects. Results No rare variants were detected in KISS1 or KISS1R in the 30 subjects with ICPP. In LIN28B, one missense change, His199Arg, was found in one subject with ICPP. However, this variant was also detected in one of the 132 controls. No variation in LIN28A was found. Conclusions We did not find any evidence that mutations in LIN28B or LIN28A would underlie ICPP. In addition, we confirmed that mutations in KISS1 and KISS1R are not a common cause for ICPP.

Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia)

Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotiana tabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP(+) to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI-MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP(+) as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.

Cardiac remodeling in Drosophila arises from changes in actin gene expression and from a contribution of lymph gland-like cells to the heart musculature

Understanding the basis of normal heart remodeling can provide insight into the plasticity of the cardiac state, and into the potential for treating diseased tissue. In Drosophila, the adult heart arises during metamorphosis from a series of events, that include the remodeling of an existing cardiac tube, the elaboration of new inflow tracts, and the addition of a layer of longitudinal muscle fibers. We have identified genes active in all these three processes, and studied their expression in order to characterize in greater detail normal cardiac remodeling. Using a Transglutaminase-lacZ transgenic line, that is expressed in the inflow tracts of the larval and adult heart, we confirm the existence of five inflow tracts in the adult structure. In addition, expression of the Actin87E actin gene is initiated in the remodeling cardiac tube, but not in the longitudinal fibers, and we have identified an Act87E promoter fragment that recapitulates this switch in expression. We also establish that the longitudinal fibers are multinucleated, characterizing these cells as specialized skeletal muscles. Furthermore, we have defined the origin of the longitudinal fibers, as a subset of lymph gland cells associated with the larval dorsal vessel. These studies underline the myriad contributors to the formation of the adult Drosophila heart, and provide new molecular insights into the development of this complex organ. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Hyper-activation of the TCP4 transcription factor in Arabidopsis thaliana accelerates multiple aspects of plant maturation

Plant organs are initiated as primordial outgrowths, and require controlled cell division and differentiation to achieve their final size and shape. Superimposed on this is another developmental program that orchestrates the switch from vegetative to reproductive to senescence stages in the life cycle. These require sequential function of heterochronic regulators. Little is known regarding the coordination between organ and organismal growth in plants. The TCP gene family encodes transcription factors that control diverse developmental traits, and a subgroup of class II TCP genes regulate leaf morphogenesis. Absence of these genes results in large, crinkly leaves due to excess division, mainly at margins. It has been suggested that these class II TCPs modulate the spatio-temporal control of differentiation in a growing leaf, rather than regulating cell proliferation per se. However, the link between class II TCP action and cell growth has not been established. As loss-of-function mutants of individual TCP genes in Arabidopsis are not very informative due to gene redundancy, we generated a transgenic line that expressed a hyper-activated form of TCP4 in its endogenous expression domain. This resulted in premature onset of maturation and decreased cell proliferation, leading to much smaller leaves, with cup-shaped lamina in extreme cases. Further, the transgenic line initiated leaves faster than wild-type and underwent precocious reproductive maturation due to a shortened adult vegetative phase. Early senescence and severe fertility defects were also observed. Thus, hyper-activation of TCP4 revealed its role in determining the timing of crucial developmental events, both at the organ and organism level.

Heterologous expression of P5CS gene in chickpea enhances salt tolerance without affecting yield

Vigna Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA was transferred to chickpea (Cicer arietinum L.) cultivar Annigeri via Agrobacterium tumefaciens mediated transformation. Following selection on hygromycin and regeneration, 60 hygromycin-resistant plants were recovered. Southern blot analysis of five fertile independent lines of T0 and T1 generation revealed single and multiple insertions of the transgene. RT-PCR and Western blot analysis of T0 and T1 progeny demonstrated that the P5CS gene is expressed and produced functional protein in chickpea. T1 transgenic lines accumulated higher amount of proline under 250 mM NaCl compared to untransformed controls. Higher accumulation of Na(+) was noticed in the older leaves but negligible accumulation in seeds of T1 transgenic lines as compared to the controls. Chlorophyll stability and electrolyte leakage indicated that proline overproduction helps in alleviating salt stress in transgenic chickpea plants. The T1 transgenics lines were grown to maturity and set normal viable seeds under continuous salinity stress (250 mM) without any reduction in plant yield in terms of seed mass.